Method Development and Validation of RP-HPLC Method for Glimepiride, Pioglitazone Hcl and Metformin Hcl in Tablets

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(1)46. IJPAR | Volume 3 | Issue 1 | Jan-Mar -2014. ISSN: 2320-2831. Available Online at: www.ijpar.com. [Research article]. Method Development and Validation of RP-HPLC Method for Glimepiride, Pioglitazone Hcl and Metformin Hcl in Tablets N.Ramathilagam*1, P.Solairaj2 *1 Department of pharmaceutical analysis, Smt Sarojini Ramulaam College of Pharmacy, Mahabubnagar, Andra pradesh, India. 2 Department of Pharmacuetical analysis, Sangaralingam Bhuvaneswari College of Pharmacy, Sivakasi, Tamilnadu, India. ABSTRACT A simple, accurate, precise and linear Isocratic RP-HPLC has been developed and subsequently simultaneous for the determination of glimepiride, pioglitazone Hcl and metformin Hcl in pharmaceutical dosage form. Kromosil C18 (150mm X4.6 mm) 5µ with flow rate of 1ml/min by using JASCO PU-1580 and UV/VIS JASCO UV-1570 at 217nm. The separation was carried out using a mobil phase consisting of the mixture of methanol and 25mM phosphate buffer (pH-2.0) in the ratio of 50 :50. The retention time for glimepiride, metformin Hcl and pioglitazine Hcl were found to be 2.24, 3.76 and 10.20 min. the correlation coefficient was found to be 0.999 for GLI, 0.9993 for PIO and 0.9997 for MET. The mean percentage recovery was found to be 98.58 (GLI), 98.30 (PIO) and 98.87 (MET). The percentage the accuracy of the drugs were found to be near to 100% representing the accuracy of the method. The proposed method was also validated and applied for the analysis of the drugs on tablet formulations. Keywords : RP-HPLC, Glimepiride (GLI), Pioglitazone Hcl (PIO) and Metformin Hcl (MET). . It is used in the treatment of type-2 diabetes INTRODUCTION mellitus and also non-insulin dependent diabetes Glimepiride is chemically 1-P-2-3 ethyl methyl-2mellitus (NIDDM). It is selectively stimulates the oxo-3pyrroline-1-carboxamido ethyl phenyl-3nuclear receptor peroxisome proliferators activated (trans-4-methyl cyclohexyl) urea, is receptor gamma (PPAR-γ). Metformin Hcl is antihyperglycemic agent. It is indicated for the chemically 1,1-dimethylbiguannide, is antilipolytic treatment of the oral therapy of type -2 diabetes effect. It act by suppressing excessive hepatic mellitus. It improves the insulin sensitivity of glucose production and improving glucose peripheral tissue and lowers blood sugar by clearance and decrease fasting plasma glucose stimulating the release of insulin by pancreatic beta levels and increase insulin-mediate glucose cells and by inducing increased activity of utilization in peripheral tissue and lowers serum intracellular insulin receptors. Pioglitazone Hcl is free fatty acid concentrations1-5. From the survey of chemically ±-5-{[4(-(5-ethyl-2-pyridinyl) ethoxy) literature, this drugs using Spectrophotometric, phenyl]methyl}-2,4 thiazodine dione mono LC/MS, IPLC, LC-ESI-MS/MS, RP-HPLC, hydrochloride, is an oral antihyperglycemic agent. HPTLC methods in single and combined with * Corresponding author: N.Ramathilagam E-mail address: [email protected].

(2) 47 N.Ramathilagam, et al / Int. J. of Pharmacy and Analytical Research Vol-3(1) 2014 [46-54]. others drugs and in human plasma. But there is no work in RP-HPLC method for the simultaneous estimation of glimepiride, pioglitazone Hcl and metformin Hcl in their combination dosage form632 . The proposed method presented here is simple, fast, accurate and precise and can be used for simultaneous determination in combination tablet dosage form. The method was validated as per ICH guidelines33-37.. INSTRUMENT Instrument used in present study was JASCO. HPLC. The pump used was JASCO PU-1580 pump. The samples were applied Kromosil c 18 5µ column with Rhedyne injector. The sample was performed using UV/VIS JASCO UV-1570 detector with flow rate 1ml/min and operated by JASCO LC –NeT II/ADC interface. The scaltech precision balance (0.001gm sensitivity) was used for weighing purpose.. 10mg of glimepiride was dissolved in 1% formic acid in acetonitrile, 10mg of pioglitazone Hcl was dissolved in methanol and 32mg of metformin Hcl was dissolved in distilled water and made upto 10ml with same for each drugs. From this solution, 1ml of solution was made up to 10ml with diluent (methanol and water in the ratio 50:50) separately and 5ml of each solution made upto 50ml with mobile phase. The working standard solution concentration was prepared to the concentration 2µg/ml, 15µg/ml and 50µg/ml.. PREPARATION OF SAMPLE SOLUTION A quantity of tablet powder equivalent to 0.04552 gm transfer to 10ml volumetric flask dissolved in few ml of diluent and made upto the volume by mobile phase. From this 1ml of solution was diluted to 20ml using the same diluent to get the concentration of 2µg of glimepiride, 15µg/ml of pioglitazone Hcl and 50µg/ml of metformin Hcl.. MATERIALS USED Glimepiride, Pioglitazone Hcl and Metformin Hcl raw materials were supplied by microlabs, Bangalore. Tripride-2 (Microlabs) was taken for study which contains 2mg of glimepiride, 15 mg of pioglitazone Hcl and 500mg of metformin Hcl. HPLC grade methanol (molychem, Mumbai), Potassium dihydrogen orthophosphate (RFCL-New Delhi), decane sulphonic acid sodium (S.D. Fine chem. Ltd), ortho phosphoric acid (Ranbaxy fine chemical. Ltd, Mumbai), HPLC grade water.. METHOD DEVELOPMENT Selection of wavelength Stock solution of 10mg/ml was prepared and further diluted to get the concentration 10µg/ml for glimepiride, pioglitazone Hcl and metformin Hcl with diluent. The wavelength was selected by scanning the above standard drugs between 200400nm. The scan results showed that reasonably maximum absorbance was recorded at 217nm. Therefore 217nm was selected as the detection wavelength for the HPLC investigation [Figure-1].. PREPARATION OF MOBILEPHASE Methanol and 25mM phosphate buffer (pH-2.0) in the ratio 50:50 were mixed, sonicated for 10 minutes and filtered through the membrane filter of micron 0.45µ.. PREPARATION OF 25Mm PHOSPHATE BUFFER 3.4gm of potassium dihydrogen ortho phosphate and 0.244gm of decane sulphonic acid sodium were transferred into 1 litre volumetric flask, dissolved by adding 100ml of distilled water and made upto to volume with distilled water, pH adjusted to 2.0 using 1% o-phosphoric acid solution.. PREPARATION OF STANDARD SOLUTIONS. METHOD The samples were applied Kromosil C18 (150 x 4.6mm) 5µ with Rhedyne injector in reverse saturation mode using methanol and 25mM phosphate buffer (pH-2.0) in the ratio of 50:50 as mobile phase with the flow rate of 1.0ml/min. The samples were performed using UV/VIS JASCO UV-1570 detector with the flow rate 1ml/min. The instrument computes accurate results with minimal time. The retention time was obtained 2.24 for GLI, 3.76 for MET and 10.20 for PIO respectively. The results of assay were reported in the Table-1 and Figure-2.. VALIDATION OF THE METHOD Accuracy Accuracy was determined by tablets samples with different known concentrations of the drugs (50%, www.ijpar.com.

(3) 48 N.Ramathilagam, et al / Int. J. of Pharmacy and Analytical Research Vol-3(1) 2014 [46-54]. 100% and 150%). Each concentration was injected in six times and the assay was performed as per the developed method. From this % recovery and the amount present (or) recovery was calculated. Results of recovery study are reported in table-2.. 4.94 µg/ml of pioglitazone Hcl and 50.70, 76.05, 101.40, 126.75 and 152.10 µg/ml of metformin Hcl were injected. Linearity of each concentration and response of ratio of each concentration was found .Linearity of each concentration was reported in table-4.1-4.3 and graph 1-3.. Precision Standard solution of GLI, PIO and MET were prepared in the same manner for the standard preparation. This solution containing 2µg/ml for glimepiride, 15 µg/ml for Pioglitazone Hcl and 50µg/ml for metformin Hcl. The repeatability was performed for six times. The result of precision was reported in table-3.. Ruggedness The above sample prepared solution and diluted to get the concentration of 2µg/ml of glimepiride, 15µg/ml of pioglitazone Hcl, and 50µg/ml of metfomin HCL of tablet sample. The 20µL was injected through column separately by two different analysis in the same HPLC system and same column. The result was reported in tablet-5.. Linearity Linearity was determination in the range of 50150% (50, 75, 100, 125 and 150%) targeted concentration of assay procedure. 2-6ml of standard solution was made upto 10ml with mobile phase in separately flask. Five series of standard solution containing 0.21, 0.32, 0.412, 0.54 and 0.64 µg/ml of glimepiride, 1.65, 2.47, 3.30, 4.12 and. Robustness The above prepared was determined by the variation of flow rate and variation of wavelength. The result was reported in table-6.. Fig.1-Overlain UV spectra of 3 standards. Fig-2- Assay chromatogram of GLI, MET and PIO. www.ijpar.com.

(4) 49 N.Ramathilagam, et al / Int. J. of Pharmacy and Analytical Research Vol-3(1) 2014 [46-54]. Graph 1- Linearity of standard glimepiride 50000 y = 67525x - 722.76 R² = 0.9991. 40000. Peak Area. 30000 20000 10000 0 0. 0.1. in µg/mL0.5 0.3Conc. 0.4. 0.2. 0.6. 0.7. Graph 2-Linearity of standard pioglitazone HCl 250000 y = 41738x + 6052.2 R² = 0.9993. 200000. Peak Area. 150000 100000 50000 0 0. 1. Conc. 3 in µg/mL4. 2. 5. 6. Graph 3- Linearity of standard metformin HCl 6000000.000 y = 35048x + 174042 R² = 0.9997. 5000000.000 4000000.000. Peak Area. 3000000.000 2000000.000 1000000.000 0.000 0. 20. 40. 60. Conc. 80 in µg/mL 100. 120. Table -1- Results of global assay Parameters Mean of peak area. Name of drug GLI MET 28462 3762411. PIO 157068. %RSD. 0.3795. 0.0132. 1.0712. Amount in mg/tablet. 1.966. 494.336. 14.746. Label claim. 2. 500. 15. % Assay. 98.45. 98.863. 98.20. www.ijpar.com. 140. 160.

(5) 50 N.Ramathilagam, et al / Int. J. of Pharmacy and Analytical Research Vol-3(1) 2014 [46-54]. Table -2- Results of global % recovery studies Different levels. 50. 100. 150. Name of drug GLI MET PIO GLI. Amount taken in µg 1 250 7.5 2. Amount found in µg 0.96 247.54 7.23 1.96. %recovery. 98.46 98.85 98.62 98.58. % of mean recovery 98.35 98.86 98.63 98.58. MET PIO GLI. 500 15 3. 494.42 14.73 2.72. 98.97 98.54 98.65. 98.93 98.58 98.68. MET PIO. 750 22.5. 741.92 21.87. 98.92 98.65. 98.93 98.59. Table -3- Results of precision studies of standard drugs Parameters Name of drug GLI MET PIO Mean of peak area 2853.52 376566.18 155874.26 Mean of retention time 2.448 3.714 9.95 SD 77.026 1836.42 3029.70 %RSD 0.2699 0.0487 1.64. Table 4.1- Results of linearity studies of standard glimepiride Concentration in µg/mL Standard peak area 0.21. 15555.168. 0.32. 21467.320. 0.43. 28551.354. 0.54. 35238.494. 0.64. 43275.802. Table 4.2- Results of linearity studies of standard pioglitazone Hcl Concentration in µg/mL Standard peak area 1.65. 71585.79. 2.47. 107404.81. 3.30. 156247.25. 4.12. 175537.78. 4.94. 212682.95. www.ijpar.com.

(6) 51 N.Ramathilagam, et al / Int. J. of Pharmacy and Analytical Research Vol-3(1) 2014 [46-54]. Table 4.3- Results of linearity studies of standard metformin Hcl Concentration in µg/mL. Standard peak area. 50.70. 1951522.2. 76.05. 2818888.7. 101.40. 3765538.2. 126.75. 4650614.5. 152.10. 5442709.8. Table-5-Ruggedness data of analyst – I and II Parameters. Analyst-I. Analyst-II. GLI. MET. PIO. GLI. MET. PIO. Mean of standard peak area. 28405.15. 3759668. 155962.3. 28599. 3765489. 157688. %RSD. 0.710. 0.13. 0.127. 0.354. 0.401. 1.353. Recovery Quantity. 1.966. 494.336. 14.746. 1.95. 496.434. 14.841. % Recovery. 98.45. 98.863. 98.20. 98.79. 98.51. 99.67. Table -6- Robustness study. Variations. Retention Time in minutes. Standard peak area. GLI. MET. PIO. GLI. MET. PIO. Floe rate at 0.8ml\min. 2.43. 4.9. 14.3. 48299.26. 4655856. 1927303. Flow rate at 1.2ml\min. 2.45. 3.68. 9.97. 29650.1. 3164563. 134215.6. Wavelength at 215 nm. 2.45. 3.68. 9.97. 38552. 3777252. 152077. Wavelength at 219 nm. 2.43. 4.07. 11.57. 33245.67. 3619664. 154009. RESULTS AND DISCUSSION The present study was aimed to developing an an accureate, precise and linear HPLC method for analysis of glimepiride, pioglitazone Hcl and metformin Hcl and in pharmaceutical dosage form as per ICH guidelines. It showed the linearity response over range 0.21-0.64µg/ml of glimepiride, 1.65-4.94µg/ml of pioglitazone Hcl and 50.70152.10µg/ml of metformin Hcl. The correlation coefficient for three drugs was found to be 0.999, 0.9993 and 0.9997. The recovery studies of these three drugs were found to be 98.58, 98.30 and. 98.87 respectively. The precision %RSD was found to be 0.0766 for glimepiride, 0.4102 for pioglitazone Hcl and 0.0612 for metformin Hcl. The ruggedness and robustness were studied with replicates standard solution of these drugs and the result was found to be acceptance criteria.. CONCLUSION The proposed method gives good, resolution between glimepiride, pioglitazone Hcl and metformin Hcl within short time (11 minutes). The validation parameters are validated and the results. www.ijpar.com.

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